A hymenoptera venom protein hhu p140 and its use

Abstract

The present application relates to a kind of HhUp140 of anthomyiidae trichogramma venom protein and its application, it relates to the field of protein engineering, its amino acid sequence is as shown in SEQ ID NO:1, the protein can be used to paralyze pest larvae, extend pest pupation period.The present application extends the pupation period of anthomyiidae trichogramma venom protein HhUp140, reduces the disease of crop by pest to some extent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, and in particular to a Megarhyssa japonica venom protein HhUp140 and application thereof. BACKGROUND

[0002] Crop pest control is closely related to agricultural food, and crop production history is accompanied by natural disasters. As early as 700 BC, there were records of insect control in China. There are more than 1600 harmful organisms that harm crops in China, of which there are 830 species of pests. At present, chemical insecticides are more commonly used, which have good insecticidal effect, but have the disadvantages of easy residue and even pollution of soil and environment.

[0003] The present application relates to the technical field of protein engineering, and in particular to a Megarhyssa japonica venom protein HhUp140 and application thereof. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application aims to provide a Megarhyssa japonica venom protein HhUp140 and application thereof, which can prolong the pupation period of pests and reduce the damage of pests to crops to some extent.

[0005] The above application objectives of the present application are achieved by the following technical solutions.

[0006] A Megarhyssa japonica venom protein HhUp140, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] The Megarhyssa japonica venom protein HhUp140 is used for prolonging the pupation period of pests.

[0008] The pests are Chilo spp. larvae.

[0009] The present application uses the HhUp140 protein obtained from the Megarhyssa japonica venom, and the research of the present application proves that the protein can make the Chilo spp. larvae twitch all over the body, the rate of back blood vessel fluctuation is accelerated, and the Chilo spp. larvae present a paralyzed state and lose the ability to move; and the protein can also inhibit the growth of pests and prolong the pupation period to some extent.

[0010] In summary, the present application has at least one of the following beneficial technical effects.

[0011] 1. The present application uses the HhUp140 protein, which can inhibit the growth of pests and prolong the pupation period to some extent.

[0012] 2. The HhUp140 protein can also paralyze pest larvae and make them lose the ability to move. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is SDS-PAGE and Western blotting figure after expression and purification of recombinant protein HhUp140-His, wherein the red arrow indicates the recombinant protein HhUp140-His;

[0014] Figure 2 is the photo of host paralysis caused by injection of HhUp140-His into the Indian meal moth larvae, wherein, 0min, video screenshot at 0min; 1min, video screenshot at 1min; 2min, video screenshot at 2min;

[0015] Figure 3 is the photo of Indian meal moth from 0 to 4 days after injection of prokaryotic expression recombinant proteins HhUp140-His, His-GFP, Csp1a-His and Venom;

[0016] Figure 4 is the effect of injection of prokaryotic expression recombinant proteins on pupation of 5th instar Indian meal moth larvae, wherein, (A) statistics of pupation rate of Indian meal moth larvae, the data in the figure is expressed as mean ± standard deviation, and the asterisk indicates significant difference between varieties (one-way ANOVA, Tukey test: ****, p<0.0001); (B) time curve of pupation of Indian meal moth larvae. Difference analysis uses Breslow test. DETAILED DESCRIPTION

[0017] The application will be further described in detail below in combination with the drawings. EMBODIMENT

[0018] Construction of recombinant plasmid

[0019] 1) Double enzyme digestion

[0020] The expression vector was cut with the restriction endonuclease BamHI, HindIII of Thermo Fisher to obtain a linear pET-28a(+) expression vector. The reaction system was added to a PCR tube and shaken to mix, incubated at 37℃ for 10 min, and then denatured at 85℃ for 10 min. The linear plasmid was detected by gel electrophoresis and recovered.

[0021] 2) Homologous recombination

[0022] Use The plus One-Step PCR Cloning Kit system (Nearshore Proteins, Suzhou) was used to insert the target fragment into the plasmid using homologous recombination. The constructed recombinant plasmid was transformed into DH5α competent cells. After heat shock transformation, 100 μL of the bacterial culture from the bottom of the tube was spread onto a plate containing Kana resistance and incubated upside down overnight. Single colonies grown on the plate were picked and placed in 1 mL of Kana resistance liquid medium and incubated at 37°C for 3 h. Using the turbid bacterial culture as a template, colony PCR was performed using 2×TaqMaster Mix (Novizan, Nanjing) reagent to verify successful plasmid transformation. 500 μL of PCR-positive bacterial culture was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing, and the remaining bacterial culture was temporarily stored at 4°C.

[0023] Expression of recombinant proteins

[0024] 1) The successfully constructed prokaryotic expression plasmid was transformed into Transetta(DE3) Chemically Competent Cell2 competent cells from Beijing TransGen Biotech Co., Ltd. After overnight culture, a single colony was picked and cultured in 1 mL of LB liquid medium with antibiotic resistance for 4 h. Then, the turbid bacterial culture was added to 200 mL of fresh, sterile LB liquid medium with Kana resistance and incubated on a shaker at 37℃ and 200 rpm for about 3-4 h to allow the OD of the bacterial culture to reach the target value. 600 The value is reduced to 0.5.

[0025] 2) Add 0.8 Mm IPTG to every 200 mL of bacterial culture and induce expression on a shaker at 120 rpm for 10 h at 24 ℃.

[0026] 3) Add the induced bacterial culture to a 100 mL centrifuge tube and centrifuge at 7000 g for 10 min at 4 °C to remove the liquid culture medium. The collected bacterial cells can be directly lysed or frozen at -80 °C for later use.

[0027] Purification of recombinant proteins

[0028] 1) Add 5 mL of B-PER to each gram of bacterial cells. TM Bacterial Protein Extraction Reagent (Thermo, USA): After the bacterial blocks were aspirated and the cells were evenly dissolved in the lysis buffer, the mixture was incubated on a shaker at 4°C for 1 hour.

[0029] 2) After complete lysis, transfer the lysate to a 2 mL centrifuge tube and centrifuge at 12000 g for 30 min at 4 °C. Collect the supernatant and discard the precipitate.

[0030] (3) Add 700 μL of cOmplete His-Tag Purification Resin (Roche Diagnostics GmbH, Germany) to the 12 mL column (Shanghai, China) and let it stand and flow out the ethanol.

[0031] 4) Add 5 mL of imidazole-free binding buffer (300 mM NaCl, 50 mM NaH2P04, pH 7.5) to the column, gently mix the resin, and let it settle before flowing out the liquid to replace the ethanol. Repeat twice.

[0032] 5) Add 10 mL of lysis buffer to the affinity column and bind overnight at 4°C on a shaker.

[0033] 6) The next day, discard the liquid in the column. Add 10 mL of rinse buffer (300 mM NaCl, 50 mM NaH2P04, 5 mM imidazole, pH 7.5) and bind for 30 min at 4°C on a shaker. Discard the liquid in the column.

[0034] 7) Wash away the bound impurities from the column with a gradient of imidazole concentrations (10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 120 mM imidazole) in rinse buffer (300 mM NaCl, 50 mM NaH2P04, pH 7.5). Follow the procedure in step (6).

[0035] 8) Add 5 mL of elution buffer (300 mM NaCl, 50 mM NaH2P04, 250 mM imidazole, pH 7.5) to the affinity column, bind for 30 min at 4°C on a shaker, collect the flow-through, and detect the band by SDS-PAGE.

[0036] Injection of recombinant protein into host

[0037] Healthy and similar-sized 5th instar Indian meal moth larvae were selected for injection. After paralyzing the larvae on ice, they were fixed in the grooves of agar plates and injected with HhUp140-His recombinant protein using a microinjector. Each Indian meal moth was injected with 1 μL of recombinant protein at a concentration of 0.9 mg / mL. GFP-His was used as a negative control, and U1-nemetoxin-Csp1a-His and Cotesia maculosa crude venom were used as positive controls.

[0038] Function of recombinant protein

[0039] Healthy and approximately 5 late instar larvae of P. interpunctella were selected to verify whether they have paralysis function by injecting 0.9 μg of recombinant protein in vitro, ten larvae were injected in each treatment, three groups of repeats were set, and the performance of the host was continuously observed. After 2 h of observation, all P. interpunctella larvae injected with HhUp140-His recombinant protein were paralyzed. Under the in vivo microscope, it can be seen that P. interpunctella injected with HhUp140-His protein has convulsions, the rate of back blood vessels fluctuation is accelerated, and presents a paralyzed state. With the change of time, the larvae injected with GFP-His protein can still move freely, while the larvae injected with HhUp140-His protein have lost the ability to move Figure 2 and Figure 3 ). In addition, the larvae injected with crude venom were completely paralyzed, and the P. interpunctella larvae injected with Csp1a-His protein showed no signs of paralysis. After 24 h, some P. interpunctella larvae injected with HhUp140-His protein recovered from paralysis and had a stress response after external stimulation, and P. interpunctella larvae injected with GFP-His control protein behaved normally and could spin silk. After 48 h, P. interpunctella larvae injected with Csp1a-His protein could not move freely and even died.

[0040] The pupation rates of P. interpunctella larvae treated in different ways were counted, and the pupation rate of larvae injected with HhUp140-His was only 53.3 ± 5.77%, which was significantly different from that of P. interpunctella injected with GFP-His control protein Figure 4 A). After three days of injection, the larvae began to pupate one after another, and from the pupation progress curve, it can be seen that there is a significant difference between P. interpunctella larvae injected with HhUp140-His and control larvae injected with GFP-His. Although the P. interpunctella larvae recovered from paralysis can still pupate, the pupation period is prolonged, which may be due to the inhibition of growth Figure 4 B).

[0041] This embodiment is only an explanation of the present application and is not a limitation of the present application. Those skilled in the art can make modifications to this embodiment without creative contribution after reading this specification, but as long as it is within the scope of the present application, it is protected by the patent law.

Claims

1. Use of a venom protein of Habrobracon hebetor HhUp140 for paralyzing pest larvae, prolonging the pupation period of pests, characterized in that, The amino acid sequence of the venom protein HhUp140 of Microplitis tuberculifer is shown as SEQ ID NO: 1; and the pest is the Lepidoptera Pyralidae Oryzaephilus indicus larvae. The amino acid sequence of the venom protein HhUp140 of Microplitis tuberculifer is shown as SEQ ID NO: 1; and the pest is the Lepidoptera Pyralidae Oryzaephilus indicus larvae. The amino acid sequence of the venom protein HhUp140

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